Method for generating induced pluripotent stem cells from keratinocytes derived from plucked hair follicles
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Example 1
The Derivation of Keratinocytes from the ORS of Plucked Hair Follicle and their Characterization
[0168]In order to generate iPSCs from hair, the present inventors followed Aasen et al.'s protocol (Aasen et al. 2008) for generating iPSCs from plucked hair follicles. Plucked hair was cultured on Matrigel-coated dishes supplemented with MEF-conditioned media for at least five days until cells proliferated out of the outer root sheath (ORS), but an insufficient number of viable and proliferative cells could be isolated. The present inventors obtained a single cell suspension of keratinocytes by plucking and selecting at least 10 single hairs with a visible bulb and intact ORS (FIG. 1A), incubating them with DMEM-supplemented with penicillin, streptomycin, HEPES and L-Glu for 24 hr, and then removing the cells from the ORS enzymatically with trypsin-EDTA (FIG. 1B). These isolated keratinocytes were seeded on inactivated 3T3 feeder cells (FIGS. 1C, D) and could be further cultured...
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Example 2
Optimization of the Reprogramming Procedure
[0170]With the purpose of developing an efficient HFKT reprogramming protocol, the present inventors used various viral vectors and growth conditions to optimize the reprogramming of common target cells, such as human foreskin fibroblasts (FIFE) and human dermal fibroblasts (HDF). The viral vectors analyzed were the pMX retroviral vectors harboring four (Klf4, Oct3 / 4, Sox2 and c-Myc) or three (Klf4, Oct3 / 4 and Sox2) reprogramming factors (Takahashi et al. 2007; Nakagawa et al. 2008), The pMSCV retroviral vector set modified by Aasen et al. (Aasen et al. 2008) and the humanized version of a single lentiviral STEMCCA vector (SEQ ID NO: 1).
TABLE 5Calibration of post-infection growth-conditionsCellsCellmaintenancesubculturingReprogrammingCell(Fresh / thawGrowthratioEfficiencysourceafter freezing)Viral vectorconditions(post infection)% + SDHFFThaw cellspMX 4FMEF feeder1:60.04% ± 0.03Fresh cells were(OSKM)layernot determinedFibronectin + ...
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Example 3
Induction of Pluripotent Stem Cells from HFKTs, and their Identity Relative to HFKT Parental Cells and hESCs
[0180]The procedure for HFKT reprogramming is illustrated in FIG. 2A. KTN and KTR keratinocytes were derived from plucked hairs of two healthy women, aged 36 and 41, designated “N” and “R”, respectively. Starting with 30,000 keratinocytes which were infected with the STEMCCA vector, ˜5-9 iPSC colonies were isolated in five independent experiments. Importantly, all the colonies that emerged following ˜30 days of incubation were true iPSCs exhibiting morphological features resembling those of hESCs (FIG. 2B). All the iPSC colonies were picked mechanically and were transferred to the MEF feeder-layer for further expansion and analysis. Their karyotypes were analyzed and were found to be normal excluding two clones, one with a lost X chromosome (45, X0) and the other with an unstable karyotype (data not shown).
[0181]Specific characterization of the HFKT-iPSCs, as compared...
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Abstract
- a. culturing isolated hair follicle keratinocytes on a layer of feeder cells, so as to generate colonies of hair follicle keratinocytes;
- b. detaching the colonies of hair follicle keratinocytes from the feeder cells so as to generate detached keratinocytes;
- c. infecting the detached keratinocytes with a virus comprising a nucleic acid molecule encoding at least one dedifferentiation factor so as to generated infected keratinocytes; and
- d. culturing the infected keratinocytes on a layer of feeder cells in a culture medium until iPS cells are formed, thereby generating iPS cells.
- Populations and uses of the iPS cells are also disclosed.
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